Acute myeloid leukemia (AML) is thought to arise when early hematopoietic stem or progenitor cells acquire mutations leading to the development of leukemia stem cells (LSCs). Failure to fully eradicate LSCs using conventional chemotherapy is responsible for disease progression and relapse, which is the main cause of death in AML. Therefore, strategies that more effectively eradicate LSCs have the potential to be highly significant and to address an urgent unmet clinical need. Recent clinical studies using a combination of the BCL-2 inhibitor venetoclax with azacitidine (ven/aza) have demonstrated remarkable results for elderly AML patients, with a high frequency of deep and durable complete remissions. These outcomes suggest that the regimen is targeting the LSC population in vivo. Our laboratory studies have shown that the central molecular mechanism driving eradication of LSCs relies upon inhibition of oxidative phosphorylation (OXPHOS). Specifically, LSCs in newly diagnosed patients utilize catabolism of amino acids as the primary fuel to drive OXPHOS. The ven/aza regimen reduces amino acid metabolism, thereby inhibiting OXPHOS and leading to LSC death. However, some AML patients are refractory or acquire resistance to the ven/aza regimen. Thus, the goal of this proposal is to elucidate the mechanisms by which LSCs become resistant to ven/aza therapy. We propose that there are at least 3 mechanisms leading to drug resistance, each involving metabolic compensation that circumvents the use of amino acid metabolism as a means to fuel OXPHOS. Our studies will involve detailed analysis of each mechanism, with the objective of defining clinical therapies designed to restore sensitivity to ven/aza and/or to target metabolic mechanisms required for LSC survival.